IL-1R1-dependent signaling coordinates epithelial regeneration in response to intestinal damage.

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1+ mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1+ mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function.

Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes.

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection.

Although flaviviruses co-opt the function of the host endoplasmic reticulum (ER) membrane protein complex (EMC) during infection, a mechanistic explanation for this observation remains unclear. Here, we show that the EMC promotes biogenesis of dengue virus (DENV) and Zika virus (ZIKV) non-structural multi-pass transmembrane proteins NS4A and NS4B, which are necessary for viral replication. The EMC binds to NS4B and colocalizes with the DENV replication organelle. Mapping analysis reveals that the two N-terminal marginally hydrophobic domains of NS4B confer EMC dependency. Furthermore, altering the hydrophobicity of these two marginally hydrophobic domains relieves NS4B's EMC dependency. We demonstrate that NS4B biogenesis, but not its stability, is reduced in EMC-depleted cells. Our data suggest that the EMC acts as a multi-pass transmembrane chaperone required for expression of at least two virally encoded proteins essential for flavivirus infection and point to a shared vulnerability during the viral life cycle that could be exploited for antiviral therapy.

A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus.

There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, which encodes the α and ß subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.

Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries.

The aminoglycoside, 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R-group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains.

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex.

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.

Identification of an Inhibitor of the Aminoglycoside 6'-N-Acetyltransferase type Ib [AAC(6')-Ib] by Glide Molecular Docking.

The aminoglycoside 6'-N-acetyltransferase type Ib, AAC(6')-Ib, confers resistance to clinically relevant aminoglycosides and is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens. An alternative to counter the action of this enzyme is the development of inhibitors. Glide is a computational strategy for rapidly docking ligands to protein sites and estimating their binding affinities. We docked a collection of 280,000 compounds from 7 sub-libraries of the Chembridge library as ligands to the aminoglycoside binding site of AAC(6')-Ib. We identified a compound, 1-[3-(2-aminoethyl)benzyl]-3-(piperidin-1-ylmethyl)pyrrolidin-3-ol (compound 1), that inhibited the acetylation of aminoglycosides in vitro with IC50 values of 39.7 and 34.9 µM when the aminoglycoside substrates assayed were kanamycin A or amikacin, respectively. The growth of an amikacin-resistant Acinetobacter baumannii clinical strain was inhibited in the presence of a combination of amikacin and compound 1.

Inhibition of aminoglycoside 6'-N-acetyltransferase type Ib-mediated amikacin resistance in Klebsiella pneumoniae by zinc and copper pyrithione.

The in vitro activity of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was inhibited by CuCl2 with a 50% inhibitory concentration (IC50) of 2.8 µM. The growth of an amikacin-resistant Klebsiella pneumoniae strain isolated from a neonate with meningitis was inhibited when amikacin was supplemented by the addition of Zn(2+) or Cu(2+) in complex with the ionophore pyrithione. Coordination complexes between cations and ionophores could be developed for their use, in combination with aminoglycosides, to treat resistant infections.

Inhibition of aminoglycoside 6'-N-acetyltransferase type Ib by zinc: reversal of amikacin resistance in Acinetobacter baumannii and Escherichia coli by a zinc ionophore.

In vitro activity of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was inhibited by ZnCl2 with a 50% inhibitory concentration (IC50) of 15 µM. Growth of Acinetobacter baumannii or Escherichia coli harboring aac(6')-Ib in cultures containing 8 µg/ml amikacin was significantly inhibited by the addition of 2 µM Zn(2+) in complex with the ionophore pyrithione (ZnPT).

Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-negatives: the Klebsiella pneumoniae Paradigm.

Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about physiology and role in virulence and antibiotic resistance of plasmids from the gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious communityand hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most of US hospital infections.

Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-Negatives: the Klebsiella pneumoniae Paradigm.

Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits, resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about the physiology of plasmids and their role in virulence and antibiotic resistance from the Gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious community- and hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most U.S. hospital infections.

Inhibitors of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] identified by in silico molecular docking.

AAC(6')-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6')-Ib and exhibited competitive inhibition against kanamycin A.

Does high knee flexion cause separation of meniscal repairs?

BACKGROUND: Previous clinical studies comparing nonrestrictive and restrictive protocols after meniscal repair have shown no difference in outcomes; however, some surgeons still limit range of motion out of concern that it will place undue stress on the repair. HYPOTHESIS: Large acute medial meniscal tears will gap during simulated open chain exercises at high flexion angles, and a repaired construct with vertical mattress sutures will not gap. STUDY DESIGN: Controlled laboratory study. METHODS: Tantalum beads were implanted in the medial menisci of 6 fresh-frozen cadaveric knees via an open posteromedial approach. Each knee underwent 10 simulated open chain flexion cycles with loading of the quadriceps and hamstrings. Testing was performed on 3 different states of the meniscus: intact, torn, and repaired. Biplanar radiographs were taken of the loaded knee in 90°, 110°, and 135° of flexion for each state. A 2.5-cm tear was created in the posteromedial meniscus and repaired with inside-out vertical mattress sutures. Displacement of pairs of beads spanning the tear was measured in all planes by use of radiostereometric analysis (RSA) with an accuracy of better than 80 µm. RESULTS: With a longitudinal tear, compression rather than gapping occurred in all 3 regions of the posterior horn of the meniscus (mean ± standard deviation for medial collateral ligament [MCL], -321 ± 320 µm; midposterior, -487 ± 256 µm; root, -318 ± 150 µm) with knee flexion. After repair, meniscal displacement returned part way to intact values in both the MCL (+55 ± 250 µm) and root region (-170 ± 123 µm) but not the midposterior region, where further compression was seen (-661 ± 278 µm). CONCLUSIONS: Acute posteromedial meniscal tears and repairs with vertical mattress sutures do not gap, but rather compress in the transverse plane at higher flexion angles when subjected to physiologic loads consistent with active, open kinetic chain range of motion rehabilitation exercises. The kinematics of the repaired meniscus more closely resemble that of the intact meniscus than that of the torn meniscus in regions adjacent to the MCL and the root but not in the midposterior region, where meniscal repair led to increased compression across the tear plane. CLINICAL RELEVANCE: This study supports the idea that nonrestrictive unresisted open chain range of motion protocols do not place undue stress on meniscal repairs.

Anatomy of the anterior interosseous innervation of the pronator quadratus: evaluation of structures at risk in the single dorsal incision wrist denervation technique.

PURPOSE: Patients with chronic wrist pain often are treated with wrist denervation, which typically involves transecting both the anterior interosseous nerve (AIN) and the posterior interosseous nerve. A single dorsal incision approach is an improvement over the more traditional multiple-incision technique. The purpose of our study was to describe the branches of the AIN to the pronator quadratus and evaluate the risk of denervation with the single dorsal incision technique. METHODS: Twelve fresh-frozen cadaver forearms were dissected. The branches of the AIN to the pronator quadratus were identified and the individual branch points were measured from the articular edge of the distal radius. Wrist denervation was then performed on each specimen through the single dorsal incision (as suggested by Berger). RESULTS: There were an average of 3 branches from the AIN to the pronator quadratus. All forearms had at least 1 branch to the pronator quadratus more proximal to the distal end of the dorsal skin incision; however, in only 2 of the forearms was the most proximal branch more than 2 cm proximal to the distal end of the dorsal skin incision. CONCLUSIONS: Wrist denervation through the recommended single dorsal incision poses a serious risk for completely denervating the pronator quadratus. Therefore the resection of the AIN must be performed close to the distal margin of the pronator quadratus.

Orthopedic injuries during Operation Enduring Freedom.

Orthopedic injuries comprise a majority of combat injuries seen in recent U.S. military conflicts. Interventions in the forward deployed area have played an important role in improving mortality rates of soldiers as well as outcome at a medical center level. A retrospective review was conducted on orthopedic injuries from Operation Enduring Freedom evaluated at Walter Reed Army Medical Center (WRAMC). Patients were grouped into one of five injury categories (open fracture, amputation, arterial injuries, neurological injuries, and soft tissue injury) with evacuation time (days from time of injury to arrival at WRAMC) and procedures performed before arrival at WRAMC evaluated. The average evacuation time for all orthopedic casualties was 7.9 days. There was an average of 2.6 procedures performed per patient before arrival at WRAMC. There was no difference in evacuation time among the injury groups. Those with only soft tissue injuries underwent fewer procedures than the other injury groups; however, there was no difference among the injury groups in terms of procedures performed. The number of procedures performed did not affect the evacuation time. Fifty-six percent of casualties required operative intervention after arrival at WRAMC. With the unavoidable evacuation time that all casualties must endure regardless of severity of the injury, early operative intervention in forward deployed medical assets, such as the forward surgical team and combat support hospital, remains a necessity for rehabilitative and reconstructive efforts of the soldiers at the medical center level.

Evaluation of orthopaedic injuries in Operation Enduring Freedom.

Orthopaedic injuries constitute a majority of the combat casualties in recent U.S. military conflicts. Orthopaedic injuries sustained in Operation Enduring Freedom from December 2001 to January 2003 that were treated in forward-deployed military medial facilities and evacuated to a U.S. army medical center were reviewed. The spectrum of injuries included open fractures, amputations, neurovascular, and soft-tissue injuries. Forty-four patients (85%) received treatment beyond local wound care prior to arrival at a military medical center. Debridement and irrigation was the most commonly performed procedure due to the contaminated nature of these combat injuries. There were no infections among patients with open fractures, and no patients with external fixators had pin tract infections. None of the open fracture patients underwent primary internal fixation or primary wound closure. The average time from injury to wound coverage of the open fracture wounds was 12 days. Two amputations were infected and were treated with revision and delayed wound closure. There were no primary amputations done at our institution due to infection or ischemia. All arterial injuries underwent urgent revascularization in a field hospital. None of the arterial repairs required revision after evacuation to a medical center. Operation Enduring Freedom has been an excellent example of how early and aggressive intervention in a forward-deployed area has a significant effect on rehabilitative and reconstructive efforts at a rear echelon tertiary care center.

Evaluation of orthopaedic injuries in Operation Enduring Freedom.

Orthopaedic injuries constitute a majority of the combat casualties in recent U.S. military conflicts. Orthopaedic injuries sustained in Operation Enduring Freedom from December 2001 to January 2003 that were treated in forward-deployed military medical facilities and evacuated to a U.S. army medical center were reviewed. The spectrum of injuries included open fractures, amputations, neurovascular, and soft-tissue injuries. Forty-four patients (85%) received treatment beyond local wound care prior to arrival at a military medical center. Debridement and irrigation was the most commonly performed procedure due to the contaminated nature of these combat injuries. There were no infections among patients with open fractures, and no patients with external fixators had pin tract infections. None of the open fracture patients underwent primary internal fixation or primary wound closure. The average time from injury to wound coverage of the open fracture wounds was 12 days. Two amputations were infected and were treated with revision and delayed wound closure. There were no primary amputations done at our institution due to infection or ischemia. All arterial injuries underwent urgent revascularization in a field hospital. None of the arterial repairs required revision after evacuation to a medical center. Operation Enduring Freedom has been an excellent example of how early and aggressive intervention in a forward-deployed area has a significant effect on rehabilitative and reconstructive efforts at a rear echelon tertiary care center.